Project Abstract The brain's circadian clock controls daily rhythms in physiology and behavior. Disruptions of circadian rhythms with shift work are associated with many human health problems. In addition, behavioral rhythms are often disrupted with aging and neurological disorders. Understanding the direct links between the circadian system and specific biological processes and behaviors will provide insights into disease susceptibility. In Drosophila melanogaster, we have a detailed understanding of the transcriptional oscillator mechanism and the specific neurons that are necessary for generating 24-hour behavioral rhythms. However, little is known about how circadian clocks communicate time-of-day information to output circuits controlling behaviors. Recent work showed that clock neurons project to neurons in the pars intercerebralis (PI), a brain region containing neurosecretory cells implicated in regulating various behaviors. PI neurons expressing DH44 neuropeptide are necessary for circadian rhythms of locomotor activity. In this proposal, I will leverage our knowledge of this circadian output circuit to understand the mechanisms by which timing signals are translated into behavioral rhythms. In Aim 1, I will identify a mechanistic basis for rhythmic output from Dh44 neurons. Since the Dh44 neurons and clock neurons have direct anatomical connections, I hypothesize that these connections provide a neural substrate that regulates the diurnal neural activity of Dh44 neurons and DH44 neuropeptide signaling. To address this, I will investigate how the circadian system regulates neural activity in and neuropeptide release from Dh44 neurons and the signaling ability of DH44 itself. By identifying a mechanism for rhythmic output at the level of DH44 signaling, we will gain insights into how this circuit encodes time-of-day information downstream of clock neurons. In Aim 2, I will determine the site of DH44 neuropeptide action. DH44 neuropeptide signals through its receptors, DH44-R1 and DH44-R2. Preliminary data show that Dh44-R1 is the receptor required for normal rest:activity rhythms. In Aim 2, I will identify the neurons where DH44-R1 is required. I will also establish functional connectivity between Dh44 and downstream neurons with in vivo imaging techniques. Together, the proposed experiments will elucidate the mechanisms by which DH44 neuropeptide signaling regulate a circadian output circuit controlling rest:activity rhythms.